Effects of Salt Stress on Growth, Antioxidant Enzyme and Phenylalanine Ammonia-Lyase Activities in Jatropha Curcas L

Effects of salt stress on growth, antioxidant enzyme and phenylalanine ammonia-lyase activities in Jatropha curcas L. seedlings

S. Gao, C. Ouyang, S. Wang, Y. Xu, L. Tang, F. Chen

Sichuan Key Laboratory of Resource Biology and Biopharmaceutical Engineering, Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, P.R. China

ABSTRACT

The eﬀects of increasing NaCl concentrations on biomass, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and phenylalanine ammonia-lyase (PAL) in Jatropha curcas L. seedlings were investigated. The fresh weights of cotyledons and radicles with increasing NaCl concentrations decreased progressively, and the fresh weight of hypocotyls reached the lowest level at NaCl concentration of 150 mmol and then increased. SOD activity in the cotyledons, hypocotyls and radicles increased gradually up to NaCl concentrations of 150, 200 and 150 mmol, respectively. The highest POD activities in the cotyledons, hypocotyls and radicles were observed at NaCl concentrations of 150, 200 and 150 mmol, respectively. CAT activity in the cotyledons, hypocotyls and radicles enhanced gradually up to 100, 200 and 150 mmol NaCl concentrations, respectively. Increased PAL activity in the hypocotyls and radicles was linearly and positively correlated with increasing NaCl concentrations, but the peak activity in the cotyledons was observed at NaCl concentration of 150 mmol. Electrophoresis analysis suggested that diﬀerent patterns in SOD and POD isoenzymes depend on NaCl concentrations and organ type, and the staining intensities of these isoforms are consistent with the changes of enzyme activity assayed in solutions.

Keywords: ROS-scavenging enzymes; in vitro embryo culture; salt tolerance; isoenzyme pattern

Salt stress in soil or water is one of the major antioxidant enzymes such as superoxide dismutase abiotic stresses especially in arid and semi-arid (SOD), peroxidase (POD) and catalase (CAT) have regions and can severely limit plant growth and been considered as a defensive team, whose com- yield. Salt stress can lead to stomatal closure, bined purpose is to protect cells from oxidative which reduces CO2 availability in the leaves and damage (Mittler 2002). Increased SOD, POD and inhibits carbon fixation, exposing chloroplasts to CAT activities are closely related to salt tolerance excessive excitation energy, which in turn could of many plants as reported in various researches increase the generation of reactive oxygen species (Rahnama and Ebrahimzadeh 2005, Azevedo Neto (ROSs) and induce oxidative stress (Parida and et al. 2006, Koca et al. 2007). These findings suggest Das 2005, Parvaiz and Satyawati 2008). that the induction of ROS-scavenging enzymes, ROSs have potential to interact with many cel- such as SOD, POD and CAT, is the most common lular components, causing significant damage to mechanism of salt tolerance for detoxifying ROS membranes and other cellular structures. However, synthesized. an elaborate and highly redundant plant ROS Jatropha curcas L., commonly known as physic network, composed of antioxidant enzymes and nut or purging nut, is a drought resistant shrub or antioxidants, is responsible for maintaining the tree belonging to the family Euphorbiaceae, which levels of ROS under tight control. In plant cell, is cultivated in many tropical and subtropical coun-

Supported by the Key Program of the State Science and Technology Commission of China, Project No. 2006BAD07A04, and by the International Cooperation Program, Project No. 2006DFB63400.

374 PLANT SOIL ENVIRON., 54, 2008 (9): 374–381 tries. Different parts of this plant could be used cluding 150 mmol NaCl and 0.5 mmol EDTA. for various purposes, such as energy source, thera- The homogenized suspension was obtained by peutic uses, fertilizer and animal feed. Jatropha centrifuging at 12 000 rpm for 10 min at 4°C. curcas is also well adapted to gravelly, sandy and The supernatant was used as assaying antioxidant saline soils where salinity might be the major enzyme and PAL. Protein content was quantified problem due to limited water supply (Openshaw by the Lowry method and bovine serum albumin 2000). Prior to this study little data known on the as the standard. changes of antioxidant enzymes and isoenzyme SOD activity was determined by the Chen and of these enzymes with respect to salt stress or Pan method (Chen and Pan 1996). One unit of SOD to possible roles in Jatropha curcas seedlings. In was defined as the enzyme activity that inhibited the present study, the effects of increasing NaCl the photoreduction of nitroblue tetrazolium to concentrations on growth, SOD, POD, CAT and blue formazan by 50%. POD activity was performed phenylalanine ammonia-lyase (PAL) in Jatropha according to the Sakharov and Aridilla method curcas L. seedlings were investigated. Moreover, (Sakharov and Aridilla 1999) with slight modifica- patterns of SOD and POD isoenzymes were also tion. One unit of POD activity was expressed as detected, to localize or demonstrate specific SOD the amount of enzyme that produced a change of and POD isoenzymes to specific organs and dif- 1.0 absorbance per min at 470 nm. CAT activity ferent NaCl concentrations. was measured following the change of absorb-

ance at 240 nm for 1 min due to H2O2. One unit of CAT activity was expressed as the amount of

MATERIAL AND METHODS enzyme needed to reduce 1 µmol of H2O2 per min (Montavon et al. 2007). SOD, POD, and CAT Mature Jatropha curcas L. seeds were harvested activities were expressed as unit per gram fresh in August 2007 from more than 10 individual wild weight (U/g fw). trees in Panzhihua, Sichuan province, China. Seeds Fresh tissues were homogenized with chilled were oven dried, selected and stored in a plastic 50 mmol Tris-HCl (pH 8.8, 1/10, w/v), supple- box (labelled, No. 20070822) and were deposited mented with 0.5 mmol EDTA and 1% polyvinyl at 4°C until use. pyrrolidone. Other steps are the same as above. The seeds were subjected to 70% ethanol for 30 s, PAL activity was measured by monitoring the re- and then to 0.1% mercuric chloride for 8 min. The action product, trans-cinnamate (Hahlbrock and seeds were rinsed several time with sterile distilled Ragg 1975). One unit of PAL activity was defined as water and soaked in sterile water for 24–36 h in the amount causing an increase of 0.01 in A290 per a culture room. Each embryo was dissected from min. PAL activity was expressed as unit per gram the seeds on a clean bench. Three embryos were fresh weight (U/g fw), as well. sown in wide-neck bottles (100 ml) containing SOD gel activity was carried out by the Beauchamp Murashige and Skoog (MS) medium (25 ml) for and Fridovich method with some modifications germination and growth. Embryos were separated (Beauchamp and Fridovich 1971). After comple- into five lots. One lot was allowed to grow on tion of electrophoresis, gels were incubated in MS medium to serve as control. The remaining 50 mmol phosphate buffer (pH 7.5) containing four lots were exposed to four concentrations of 28 µmol riboflavin, 28 mmol N,N,N,N-tetramethyl NaCl: 50, 100, 150 and 200 mmol. MS medium ethylenediamine (TEMED) for 30 min under dark pH was adjusted to 5.8 ± 0.1 prior to autoclaving conditions, followed by washing in distilled water at 121 ± 2°C for 15 min, with 30 g/l sucrose and for 1 min and incubation in the same buffer con- 6 g/l agar powder. Germination experiment was taining 2.45 mmol nitroblue tetrazolium (NBT) carried out at 30°C in a greenhouse. Rotten and for 20 to 30 min exposed to light at room tem- contaminated embryos were removed promptly. perature. Isoenzymes appeared as colorless bands When the cotyledons of seedlings had developed, on a purple background. POD isoenzyme activity cotyledons, hypocotyls and radicles were washed was determined by the procedure described by with distilled water, blotted and immediately frozen Ros Barcelo (1987). Gels, rinsed in water, were in liquid nitrogen or stored at –80°C for analysis. incubated in a solution composed of 0.06% (v/v) Three sets of seedlings were analyzed for each NaCl H2O2, 0.1% (w/v) benzidine and 0.1% (v/v) acetic concentration, with 15 embryos per set. acid at room temperature till brown bands appear, Fresh tissues were homogenized with 50 mmol and the reaction was stopped by rinsing the gels sodium phosphate buffer (pH 7.0, 1/10, w/v) in- with deionized water.

PLANT SOIL ENVIRON., 54, 2008 (9): 374–381 375 All treatments were arranged in a completely parameters were consistent with the changes in randomized design with three replicates. All data the fresh weight of cotyledons, hypocotyls and were expressed as means ± SD. Statistical signifi- radicles at all tested NaCl concentrations. Our cance was evaluated with the Student’s t-test, and findings suggested that different changes are related differences were considered significant if P values to the effects of different NaCl concentrations on were 0.05. different organs of seedlings. SOD is one of several important antioxidant enzymes with the ability to repair oxidation dam- RESULTS AND DISCUSSION age caused by ROS. Thus, SOD is considered a key enzyme for maintaining normal physiological Soil salinity is a prevalent abiotic stress for plants. conditions and coping with oxidantive stress in the Growth inhibition is a common response to salin- regulation of intracellular levels of ROS (Mittler ity and plant growth is one of the most important 2002). Effects of NaCl on SOD activity in the coty- agricultural indices of salt stress tolerance as indi- ledons, hypocotyls and radicles were shown in cated by different studies (Parida and Das 2005). Figure 2. SOD activity in the cotyledons increased In order to define salt stress tolerance or sensitiv- significantly with increasing NaCl concentra- ity of seedlings, the effects of NaCl treatment on tions compared to the control, and the maximal fresh weights of the cotyledons, hypocotyls and levels increased by 70.8% at NaCl concentration radicles were tested (Figure 1). The fresh weight of 150 mmol. Similarly, the highest SOD activi- of cotyledons and radicles showed no significant ties in the hypocotyls and radicles increased by changes at NaCl concentration of 50 mmol, but 86.8% and 72.8% at NaCl concentrations of 200 and they decreased progressively with increasing NaCl 150 mmol compared to the control, respectively. concentration. The fresh weights of hypocotyls Many studies found a positive correlation be- decreased gradually up to 150 mmol NaCl con- tween salt stress and the abundance of SOD in centration then increased at NaCl concentration plants cells (Badawi et al. 2004, Cavalcanti et al. of 200 mmol. The cotyledon area and height of 2007). Results in published reports also indicate seedlings were the most sensitive to salt stress, that SOD overexpression may be involved in the especially under higher NaCl concentrations, which increase of stress protection observed in some led to below medium of seedlings more sensitive to transgenic plants (Yiu and Tseng 2005, Tseng et salt stress than above medium. It is worth noting al. 2007). A significant increase of SOD activity in that at NaCl concentration of 200 mmol the radicles our study may be either necessary for the increased of seedlings did not develop, and the hypocotyls production of ROS or be a defensive mechanism became rough (data not shown). These growth developed by Jatropha curcas seedlings against

120 Cotyledons�100%�=�71.4�mg�FWfw (%) Hypocotyls�100%�=�82.5�mg�FWfw Radicles�100%=�73.8�mg�FWfw 90

0 0 50 100 150 200 NaCl concentrations (mmol)

Figure 1. Effects of NaCl stress on biomass of the cotyledons, hypocotyls and radicles in Jatropha curcas L. seedlings. The values and standard errors (vertical bars) of three replicates are shown

376 PLANT SOIL ENVIRON., 54, 2008 (9): 374–381 200 Cotyledons�100%�=�108.3�U/g�fw Hypocotyls�100%�=�67.6�U/g�fw (%) Radicles�100%�=�79.4�U/g�fw

150

100

0 0 50 100 150 200 NaCl concentrations (mmol)

Figure 2. Effects of NaCl stress on superoxide dismutase (SOD) activity in the cotyledons, hypocotyls and radicles of Jatropha curcas L. seedlings. The values and standard errors (vertical bars) of three replicates are shown salt stress. Patterns of gel electrophoresis analy- mechanism of Jatropha curcas seedlings against sis suggested that at least three SOD isoenzyme salt stress. Moreover, the staining intensities of bands in the cotyledons, hypocotyls and radicles these isoenzymes showed a similar change tread are detected, respectively (Figure 3). The staining compared to the quantitative changes assaying in intensities of isoenzyme I and II in the cotyledons, solutions (Figures 2 and 3). These findings suggest hypocotyls and radicles were induced significantly that different SOD isoenzymes might be regulated with increasing NaCl concentrations, but that of by salt stress and be involved in distinct physi- isoenzyme III was minimally changed in response ological processes. The present findings could also to NaCl concentrations. Thus, the changes of be used as a basis for elucidating the mechanisms staining intensities of these isoenzymes strongly how the levels of SOD transcripts are induced in depended on NaCl concentrations and plant or- response to salt stress. gans. Increased activity of SOD isozymes in dif- POD is widely distributed in higher plants where ferent organs could contribute to the tolerance it is involved in various processes, including lig-

Figure 3. Patterns of SOD isoenzymes in the cotyledons, hypocotyls and radicles of Jatropha curcas L. seedlings: (A) patterns of SOD isoenzymes in the cotyledons; (B) patterns of SOD isoenzymes in the hypocotyls; (C) patterns of SOD isoenzymes in the radicles. Lanes from 1 to 5 were 0, 50, 100, 150 and 200 mmol, respectively. About 25 µl extract from each sample was loaded

PLANT SOIL ENVIRON., 54, 2008 (9): 374–381 377 250

Cotyledons�100%�=�165.9�U/g�fw (%) Hypocotyls�100%�=�150.3�U/g�fw 200 Radicles�100%�=�246.6�U/g�fw

150

100

0 0 50 100 150 200 NaCl concentrations (mmol)

Figure 4. Effects of NaCl stress on peroxidase (POD) activity in the cotyledons, hypocotyls and radicles of Jat- ropha curcas L. seedlings. The values and standard errors (vertical bars) of three replicates are shown nification, auxin metabolism, salt tolerance and salt concentrations (Figure 4). Considering those heavy metal stress (Passardi et al. 2005). Therefore, in relation to growth parameters and the changes POD has often served as a parameter of metabo- in SOD activity (Figures 1 and 2), these results lism activity during growth alterations and en- suggest a possibility that increased POD and SOD vironmental stress conditions. Effects of NaCl activities might enable plants to protect themselves on POD activity in the cotyledons, hypocotyls against salt stress. POD isoenzymes are known to and radicles were shown in Figure 4. In general, occur in a variety of plant tissues. The expression POD activity in the cotyledons, hypocotyls and pattern of these isoenzymes varies in different radicles increased significantly with rising NaCl tissues of healthy plants and is developmentally concentrations up to 150, 200 and 150 mmol, re- regulated or influenced by environmental factors spectively. Thus, the activities increased most by (Passardi et al. 2005). To determine whether there 52%, 122.2% and 62.2% compared to the control. were developmental or salt stress-mediated dif- Increased POD activity in the hypocotyls was ferences among individual POD isoenzymes, POD more pronounced as compared to those in the activity assays were also performed by activity cotyledons and radicles, especially under higher staining (Figure 5). On the activity gels, at least

Figure 5. Patterns of peroxidase (POD) isoenzymes in the cotyledons, hypocotyls and radicles of Jatropha curcas L. seedlings: (A) patterns of POD isoenzymes in the cotyledons; (B) patterns of POD isoenzymes in the hypocotyls; (C) patterns of POD isoenzymes in the radicles. Lanes from 1 to 5 were 0, 50, 100, 150 and 200 mmol, respectively. About 10 µl extract from each sample was loaded

378 PLANT SOIL ENVIRON., 54, 2008 (9): 374–381 seven POD isoenzyme bands in the cotyledons activity in Cassia angustifolia L. (Agarwal and were visualized (Figure 5A), whereas six POD Pandey 2004), maize (Azevedo Neto et al. 2006) isoenzyme bands in the hypocotyls and radicles and Sesamum indicum (Koca et al. 2007) differ- were observed (Figure 5B and C). These isoenzymes ing in salt tolerance were found. The changes showed different staining intensities with individual in CAT activity may depend on the species, the NaCl concentrations and organs of seedlings. The development and metabolic state of the plant, as regulatory mechanism of POD isoenzymes against well as on the duration and intensity of the stress salt stress might be complicated, and the relation- (Chaparzadeh et al. 2004). This study lends further ship of the genes to these enzymes requires further support to those findings. Increased CAT activity analysis. In addition, the changes in the staining in Jatropha curcas seedlings was observed, espe- intensities of these isoenzymes showed a similar cially in the radicles, which suggested the exist- tread compared to the quantitative changes of POD ence of an effective ROS-scavenging mechanism activity assaying in solutions (Figures 4 and 5). because radicles are the first organs which come These results suggest that the increased POD activ- in contact with salt and are thus thought to play ity could contribute to the antioxidant mechanism a critical role in plant salt tolerance. Therefore, of Jatropha curcas seedlings against higher NaCl our findings showed that increased CAT activity concentrations stress. coordinated with the changes of SOD and POD CAT, which is involved in the degradation of activities plays an important protective role in hydrogen peroxide into water and oxygen, is the the ROS-scavenging process and the active in- most effective antioxidant enzymes in preventing volvement of these enzymes are related, at least oxidative damage (Willekens et al. 1995, Mittler in part, to salt-induced oxidative stress tolerance 2002). Effects of NaCl on CAT activity in the coty- in Jatropha curcas seedlings. ledons, hypocotyls and radicles were shown in PAL, a key enzyme in the phenylpropanoid path- Figure 6. CAT activity in the cotyledons increased way, is involved in the defence response of plant gradually with increasing NaCl concentrations up cells. Thus, PAL has been generally recognized as to 100 mmol, and the highest activity increased a marker of environmental stress in different plant by 75.4% compared to the control. CAT activ- species (MacDonald and D’Cunha 2007). Effects of ity in the hypocotyls enhanced significantly and NaCl on PAL activity in the cotyledons, hypocotyls reached the maximal level at NaCl concentration and radicles were shown in Figure 7. PAL activity of 200 mmol, increasing by 246.9%. Similarly, the in the cotyledons increased gradually up to NaCl peak CAT activity in the radicles was observed at concentration of 150 mmol, and the highest activity NaCl concentration of 150 mmol, increasing by increased by 117.2% compared to the control. A sig- 588.1%. Similar to our findings, increased CAT nificantly increasing PAL activity in the hypocotyls

800 Cotyledons�100%�=�45.9�U/g�fw (%) Hypocotyls�100%�=�22.8�U/g�fw Radicles�100%�=�9.2�U/g�fw 600

400

200

0 0 50 100 150 200 NaCl concentrations (mmol)

Figure 6. Effects of NaCl stress on catalase (CAT) activity in the cotyledons, hypocotyls and radicles of Jatropha curcas L. seedlings. The values and standard errors (vertical bars) of three replicates are shown

PLANT SOIL ENVIRON., 54, 2008 (9): 374–381 379 Cotyledons�100%�=�98.8�U/g�fw 400 Hypocotyls�100%�=�21.9�U/g�fw

(%) Radicles�100%�=�26.3�U/g�fw

300

200

100

0 0 50 100 150 200 NaCl concentrations (mmol)

Figure 7. Effects of NaCl stress on phenylalanine ammonia-lyase (PAL) activity in the cotyledons, hypocotyls and radicles of Jatropha curcas L. seedlings. The values and standard errors (vertical bars) of three replicates are shown was observed at all tested NaCl concentrations, and Jatropha curcas plant, and may provide useful the highest activity increased by 294.5% at NaCl bioassays that help to devise strategies for reducing concentration of 200 mmol. Similarly, the highest the risks associated with salinity toxicity, especially PAL activity in the radicles was observed at NaCl when they are combined with field experiments. concentration of 150 mmol. In a few investiga- Further experiments are necessary to understand tions, PAL activity was induced by various biotic the induction and regulation of these enzymes as and abiotic stresses, such as wounding, chilling, well as the regulation mechanism of metabolism heavy metal and infection by viruses, bacteria or in Jatropha curcas plants against salt stress; this fungi (MacDonald and D’Cunha 2007). Now the experiment is going on in our laboratory. responses of PAL activity of Jatropha curcas plants grown under higher NaCl concentrations are also known. Increased PAL activity could be a response Acknowledgements to the cellular damage provoked by higher NaCl concentrations. So it seems that in Jatropha curcas The authors sincerely thank Mr. Thomas Keeling L. seedlings the enhancement of PAL activity could for critical reading of the manuscript. be related to the implication of this enzyme in the plant response to salt stress. Moreover, our results suggest that PAL activity and the changes in PAL REFERENCES activity strongly depend on the organ of seedlings and NaCl concentrations. Agarwal S., Pandey V. (2004): Antioxidant enzyme Our findings show significant differences between responses to NaCl stress in Cassia angustifolia. Biol. responses of antioxidant defence enzymes and dif- Plant., 48: 555–560. ferent plant organs when exposed to increasing Azevedo Neto A.D., Prico J.T., Eneas-Filho J., Braga NaCl concentrations. The presented results also de Abreu C.E., Gomes-Filho E. (2006): Effect of salt clearly indicate that Jatropha curcas L. seedlings stress on antioxidative enzymes and lipid peroxidation tolerate NaCl up to a concentration of 150 mmol, in leaves and roots of salt-tolerant and salt-sensitive and higher concentrations cause eco-toxicity to maize genotypes. Environ. Exp. Bot., 56: 235–241. the plant. Increased SOD, POD, CAT and PAL Badawi G.H., Yamauchi Y., Shimada E., Sasaki R., Ka- in Jatropha curcas seedlings suggest the toler- wano N., Tanaka K., Tanaka K. (2004): Enhanced ance capacity of the plant to protect themselves tolerance to salt stress and water deficit by overex- from oxidative damage. Such a test may be useful pressing superoxide dismutase in tobacco (Nicotiana for elucidating the salt tolerance mechanisms of tabacum) chloroplasts. Plant Sci., 166: 919–928.

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Corresponding author:

Dr. Fang Chen, Sichuan University, College of Life Sciences, Ministry of Education, Key Laboratory of Bio-resources and Eco-environment, 610064 Chengdu, P.R. China phone: + 862 885 417 281, fax: + 862 885 417 281, e-mail: [email protected]

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